Background: Evidence continues to mount concerning the importance of the enteric nervous system (ENS) in controlling numerous intestinal functions in addition to motility and epithelial functions. Nevertheless, little is known concerning the direct participation of the ENS in the inflammatory response of the gut during infectious or inflammatory insults. In the present study we analyzed the ENS response to bacterial lipopolysaccharide, in particular the production of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF- α ). Methods: TNF- α expression (measured by qPCR, quantitative Polymerase Chain Reaction) and production (measured by ELISA) were measured in human longitudinal muscle-myenteric plexus (LMMP) and rat ENS primary cultures (rENSpc). They were either treated or not treated with lipopolysaccharide (LPS) in the presence or not of electrical field stimulation (EFS). Activation of extracellular signal-regulated kinase (ERK) and 5 ? -adenosine monophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Western blot analysis. Their implications were studied using specific inhibitors (U0126, mitogen-activated protein kinase kinase, MEK, inhibitor and C compound, AMPK inhibitor). We also analyzed toll-like receptor 2 (TLR2) expression and interleukin-6 (IL-6) production after LPS treatment simultaneously with EFS or TNF- α -neutralizing antibody. Results: Treatment of human LMMP or rENSpc with LPS induced an increase in TNF- α production. Activation of the ENS by EFS significantly inhibited TNF- α production. This regulation occurred at the transcriptional level. Signaling analyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpc neurons. Both U0126 and C compound almost completely prevented LPS-induced TNF- α production. In the presence of LPS, EFS inhibited the ERK and AMPK pathways. In addition, we demonstrated using TNF- α -neutralizing antibody that LPS-induced TNF- α production increased TLR2 expression and reduced IL-6 production. Conclusions: Our results show that LPS induced TNF- α production by enteric neurons through activation of the canonical ERK pathway and also in an AMPK-dependent manner. ENS activation through the inhibition of these pathways decreased TNF- α production, thereby modulating the inflammatory response induced by endotoxin.